From multiple column retention

Since ProteinTrawler records the retention time of each protein mass, it is a simple endeavor to maintain chromatographic conditions, split the flow that exits the LC column with a small portion set to the mass spectrometer to monitor for assurance that there were no changes in the retention time that would hinder the pooling of fractions from multiple runs, and to facilitate the determination of which fractions contained the desired proteins. In our experimental setup, the flow was split after the column with 25% of the flow going to the mass spectrometer while the remaining diverted to an HP1100 fraction collector. The fraction collector was used to collect fractions at 1.0-minute intervals. 

HPLC Purification Steps. Reverse phase (R HPLC Step A Filtered samples were fractionated on a Supelcosil LC-18DB column with a Pelliguard guard column (Supelco) at ambient temperature on a Model 840 nquid chromatograph with autosampler (Waters). The sample was injected onto the column and eluted with a concave gradient Waters curve 7) over one hour at 1.0 ml/min beginning with 10% acetonitrile (0.1% v/v TFA) and 90% aqueous 0.1% IPA, and ending with 60% acetonitrile (0.1% v/v TFA and 40% aqueous 0.1% TFA. The eluant was monitored spectrophotometrically at 214 nm and by fluorescence (excitation = 230 nm, emission >300 nm). Fractions were collected over 1 min intervals, with both the autosampler and fraction collector cooled to 0-5 C. Fractions with the same retention times from multiple runs were pooled in the fraction collector and dried in the Speed-Vac concentrator ( . 500 BR-SOG/run). 

Not only variations in the pressure at constant temperature influence column-to-column retention data the role of the column hold-up volume as well as the mass of stationary phase present in the column is also important. The net retention volume caleulated from the adjusted retention volume corrects for the column hold-up volume (see Table 1.2). The specific retention volume corrects for the different amount of stationary phase present in individual colunms by referencing the net retention volume to unit mass of stationary phase. Further correction to a standard temperature of 0°C is discouraged [16-19]. Such calculations to a standard temperature significantly distort the actual relationship between the retention volumes measured at different temperatures. Specific retention volumes exhibit less variability between laboratories than other absolute measures of retention. They are not sufficiently accurate for solute identification purposes, however, owing to the accumulation of multiple experimental errors in their determination. Relative retention measurements, such as the retention index scale (section 2.4.4) are generally used for this purpose. The specific retention volume is commonly used in the determination of physicochemical properties by gas chromatography (see section 1.4.2). 

With the program and derived mathematics, the scientist can calculate the retention times and peak widths for any set of solutes, run under any conditions ranging from Isocratlc to complex gradients with multiple columns. Given this mathematical tool, the problem of determining which chromatographic conditions will achieve the desired separation, within any constraints, becomes the next problem to be approached. The solution to such an "optimization" problem Is not easy, however, the necessary universal mathematical tools are now available for researchers In this area to develop approaches to optimization strategies. 

Figure 7 Enantiomer separation of methyl 2-chloropropanoate by gas chromatography on heptakis(3-0-trifluoroacetyl-2,6-di-0-/>pentyl)-)5-cyclodextrin (23) (undiluted) at three temperatures. Column 20 m x 0.25 mm (i.d.) nondeactivated fused-silica capillary. (From Berthod A, Li W, and Armstrong DW (1992) Multiple enantioselective retention mechanisms on derivatized cyclodextrin gas chromatography chiral stationary phases. Analytical Chemistry 64 873-879.)...

The aids to chromatography include a) resolution calculations on chromatograms of standard mixtures to monitor column performance, b) calculation of Kovats retention index for help in identifying peaks, and (c) multiple point calibration curves for improved quantitation. The file searching routines access two sets of data. Information (such as molecular formula, molecular weight) is stored on 3100 compounds from the Arctander data( ). This allows a quick computer search through the data which is difficult... 

Once introduced into the column, FAMEs with different carbon chain length and saturation levels move through the column at different rates and elute from the end of the column sequentially. Fatty acid standards (Nu Chek Prep or Matreya) are required to obtain the retention time for individual fatty acids in GC analysis, so that fatty acids in samples can be identified by comparing their retention times with those of the standards. In a GC run of multiple samples, standards should be analyzed prior to, during, and at the end of the sample analysis to compensate for shifts in retention times. 

Table 3 (73) compares the retention coefficients for synthetic peptides from various sources. To ensure comparability, the data has been standardized with respect to lysine and assigned a value of 100. The table shows that there are discrepancies between the results obtained using different chromatographic systems. Predictions of retention times should therefore be made using chromatographic systems similar to those used to calculate the retention coefficients for the amino acids. Casal et al. (75a) have made a comparative study of the prediction of the retention behavior of small peptides in several columns by using partial least squares and multiple linear regression analysis. 

The modem GC data system will produce a report of peaks detected with the retention time, peak area, and peak height. In order to identify the analytes of interest and quantify the data, a series of calibration standards are required to be analyzed followed by samples. The calibration standards will identify retention times for analytes, surrogates, and internal standards. With the exception of MS analysis, compounds are identified in chromatograms based solely on their retention time. Positive confirmation can be done by analyzing the same sample extract on a different type (polarity) of GC column. If the compound is detected at the same concentration from both GC columns, then the data can be reported (e.g., US EPA Method 8081—OC Pesticides—requires analysis on a DB-5 column with confirmatory analysis on a DB-17 column). For MS analysis, multiple ion chromatograms... 

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